Chlamydia trachomatis is the leading cause of preventable blindness and a major cause of sexually transmitted disease. A common feature of infection is a chronic damaging inflammatory response for which the molecular pathogenesis is not understood, It has been proposed that chlamydiae have cytotoxic activity that contributes to this pathology, but a toxin has not been identified. We have identified a cytotoxin associated with certain chlamydial strains and believe that the toxin represents a novel virulence factor that is important in the pathophysiology of chlamydial diseases.C. trachomatis isolates are separated into biovariants depending on the disease they cause. The trachoma biovars produce localized infection of the eye or genital mucosa. Infections with these strains are a major cause of STD and are characterized by persistent infection of mucosal surfaces resulting in chronic inflammatory disease. In women these infections are manifested as pelvic inflammatory disease whose sequelae can produce tubal blockage and infertility as well as tubal pregnancy. In contrast, the non-trachoma strains cause lymphogranuloma venereum (LGV) an invasive STD. LGV strains transiently infect the genital mucosa but then rapidly disseminate to the draining lymph nodes where the parasite infects monocytes and granuloma formation. An understanding of the molecular basis for these marked differences in virulence and pathogenesis are likely key to the identification of new parasite molecules that can be targeted for novel intervention strategies against chlamydial disease. The genomes of a trachoma-STD and a LGV biovarinat have recently been sequenced and annotated. Comparative genomic analysis showed remarkable similarity of the small1-1.2 million base pair genomes of each biovarinat despite their marked differences in tissue tropism and disease manifestation. However, a single gene family (CT 166-169) with homology to the large clostridial cytotoxin toxin B was found in the trachoma-STD biovariants genome which was not present in the LGV genome. It is therefore possible that this putative chlamydial toxin plays a critical role in the virulence and pathogenesis of the trachoma-STD biovariants. We have therefore focused this project on studies to define whether the trachoma-STD variants express a biologically active toxin and if so what role the toxin plays in the pathogenesis of disease caused by these medically important pathogens.Our first goal was to document that trachoma-STD biovariants and not LGV strains express an active toxin. We found that inoculation of HeLa 229 cells with a high multiplicity of infection of a trachoma-STD strain makes a replication-independent cytotoxic activity that produces morphological and cytoskeletal changes in epithelial cells that are indistinguishable from those mediated by clostridial toxin B. Cytotoxin gene transcripts were detected in chlamydial-infected cells and a protein with expected molecular mass was present in infected cells during the period of cytotoxicity. The protein was present transiently infected cells during the period of cytotoxicity. Together, these results provide compelling evidence for a chlamydial cytotoxin for epithelial cells and imply that the cytotoxin is present in the elementary bodies and is delivered to host cells very early during infection. We hypothesize that the cytotoxin is a virulence factor that contributes to the pathogenesis of C. trachomatis disease.